1. Field of the Invention
The present invention relates to a polypeptide that is modified to have homoserine acetyltransferase activity, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, a microorganism that is transformed with the recombinant vector, and a method for producing O-acetyl homoserine using the microorganism.
2. Description of the Related Art
Methionine is one of the essential amino acids in the body, and has been widely used as an animal feed and food additive, as well as a component of medical aqueous solutions and other raw materials for medicinal products. Methionine acts as a precursor of choline (lecithin) and creatine, and is also used as a raw material for the synthesis of cysteine and taurine. In addition, it functions as a sulfur donor.
S-adenosyl-methionine is derived from L-methionine and serves as a methyl donor in the body, and it is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) is/are also found to prevent lipid accumulation in the liver and arteries and to be effective for the treatment of depression, inflammation, liver diseases and muscle pain.
Methionine can be chemically or biologically synthesized to be used in animal feed, food and medicines.
In the chemical synthesis, L-methionine is mostly produced by hydrolysis of 5-(β-methylmercaptoethyl)hydantoin. However, the chemically synthesized methionine has a disadvantage of only being produced as a mixed form of L-type and D-type.
With regard to biological synthesis of L-methionine, U.S. Patent Publication No. US2005/0054060A1 describes a method of synthesizing homocysteine or methionine directly using H2S or CH3SH, while not using cysteine, by modifying cystathionine synthase for the preparation of microorganisms. In this method, modified cystathionine synthase is directly introduced into cells to synthesize methionine according to intracellular methionine synthesizing process. However, there are practical problems in that this method produces only a small amount of methionine because of inhibitory actions of synthesized methionine resulting from using intracellular methionine metabolic pathways, and H2S or CH3SH also causes cytotoxicity.
To solve these problems, the present inventors had developed a two-step process of converting L-methionine precursor into L-methionine by enzyme reaction (PCT/KR2007/003650). This two-step process can solve the above problems of cytotoxicity of H2S or CH3SH and metabolic process inhibition by produced L-methionine. Moreover, this process is characterized in that it is very efficient to produce only L-methionine selectively, and not a mixed form of D-methionine and L-methionine.
In this two-step process, O-succinyl homoserine and O-acetyl homoserine can be used as the methionine precursor. During conversion reaction of methionine, O-acetyl homoserine is advantageous over O-succinyl homoserine in terms of production yield of precursor to methionine ratio. Specifically, 0.91 mole of methionine can be produced from 1 mole of O-acetyl homoserine whereas only 0.67 mole of methionine can be produced from 1 mole of O-succinyl homoserine. Thus, production cost of the final product methionine can be reduced by using O-acetyl homoserine as the methionine precursor, and high production yield of O-acetyl homoserine is a crucial factor for the mass-production of methionine.
Meanwhile, use of the O-acetyl homoserine or O-succinyl homoserine as the methionine precursor depends on the type of microorganisms. In detail, microorganisms belonging to the genus Escherichia, Enterobacteria, Salmonella, and Bacillus produce O-succinyl-homoserine from homoserine and succinyl-coA by L-homoserine O-succinyltransferase (Biochemistry. 1999 Oct. 26; 38(43): 14416-23), and microorganisms belonging to the genus Corynebacterium, Leptospira, Deinococcus, Pseudomonas, and Mycobacterium produces O-acetyl-homoserine from homoserine and acetyl-coA by L-homoserine O-acetyltransferase (Journal of Bacteriology, March 2002, p. 1277-1286).
Therefore, expression of O-acetyl homoserine transferase by introduction of metX, a foreign gene, is required for the biosynthesis of O-acetyl homoserine using microorganisms of the genus Escherichia which are used to produce recombinant proteins for experimental and industrial purposes. However, there are problems related to negative attitudes of consumers toward introduction of foreign genes into microorganisms used for the production of food products, and proving safety of introduction of foreign genes.
Accordingly, the present inventors have made efforts to prepare a strain of the genus Escherichia that produces O-acetyl homoserine advantageous in terms of the production yield without introduction of foreign genes. As a result, they found that homoserine succinyltransferase activity can be converted into homoserine acetyltransferase activity by using a modified polypeptide prepared by substituting glutamic acid for amino acid at position 111 of O-succinyl homoserine transferase which is from E. coli, thereby completing the present invention.